C57BL/6 mice were purchased from Jackson Laboratory and bred onsite to generate animals for experimentation. Age and gender-matched 8–10 week old mice were used. The USA300 MRSA strain was grown overnight in TS broth (37°C, 180 RPM). Bacteria was then subcultured in TSB for 3h at 37°, 180 RPM. Bacteria was washed with PBS and adjusted to 1×10^7 CFU/mL. Three mice were injected intraperitoneally with 300μL. After 1h, cells were collected by peritoneal lavage. Red blood cells were lysed with BD Pharm Lyse Lysis Buffer and cells were stained with antibodies to identify macrophages. Infected macrophages were sorted (SY3200 cell sorter, HAPS1 (100um)) and samples were pooled from the three mice for scRNA-Seq analysis (Figure S6A ). Cells were captured using the 10X Chromium platform and processed according to manufacturer recommendations. All animal studies were performed as per an NYU Grossman School of Medicine Institutional Animal Care and Use Committee (IACUC) approved protocol to the Torres Lab.
Pharm lyse lysis buffer
Manufactured by BD
Sourced in United States
BD Pharm Lyse lysis buffer is a solution used to lyse or break down cells and release their contents, including proteins and nucleic acids, for further analysis. It is designed for use in various laboratory applications that require cell lysis, such as protein extraction and nucleic acid purification.
Automatically generated - may contain errors
Lab products found in correlation
C57bl 6 mice, by Jackson ImmunoResearch (2 mentions)
Collagenase type 1, by Merck Group (1 mentions)
Dnase type 1, by Merck Group (1 mentions)
Facsaria 2 system, by BD (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
Pe rat anti mouse cd8a, by BD (1 mentions)
Apc cy 7 rat anti mouse cd4, by BD (1 mentions)
Fitc anti mouse cd19, by BioLegend (1 mentions)
Mouse fcr blocking reagent, by Miltenyi Biotec (1 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (1 mentions)
Trypan blue stain, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa, by BD (1 mentions)
Purified anti mouse cd16 32 antibody, by BioLegend (1 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
10 protocols using pharm lyse lysis buffer
1
Isolation of Infected Macrophages from MRSA-Challenged Mice
2
Murine Lung Infection and Analysis
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Mice were infected oropharyngeally with P. aeruginosa PA14wt (3x106 CFU in 50ul PBS) or just PBS for 3hrs. Euthanized mouse lungs were perfused with 5 mL of ice-cold PBS injected through the right ventricle of the heart. Perfused lungs were removed and placed in RPMI on ice until processing. Lungs were minced and placed in digestion buffer of 1 mg/mL collagenase type I + 60 U DNase type I (Sigma-Aldrich) in DPBS Ca2+ Mg2+ (HyClone). Lungs were digested for 30 min at 37°C while shaking, and passed through a 70-μm filter cell strainer. Digested contents of the lungs were plated on LB agar plates to determine recovery CFU. Cells were washed (in RPMI, 1500rpm, 4°C) and residual red blood cells were lysed using 1X BD Pharm Lyse lysis buffer (BD Biosciences), for 2 min, and counted. Cells were washed and subsequently blocked using mouse Fc Block (1:200, 10 min, 4°C). Staining for flow cytometry was performed as mentioned above.
3
HFRS Patient Immune Cell Analysis
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The research was carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Ethical permits were obtained from the Swedish Ethical review authority (No: 2016/53-31, 04-113M, 07-162M and 2014/233) and all samples were collected after receiving informed consent from patient or patient’s guardian. Briefly, blood was collected in EDTA tubes and PBMCs were isolated using a Ficoll-Paque density gradient centrifugation. Blood from HFRS patients were collected 6–10 days after disease onset. Tonsillar cell suspensions were prepared by tissue homogenizing in RPMI-1640 medium and passed through a 70 µm cell strainer. Red blood cells were lysed using BD PharmLyse lysis buffer according to manufacturer’s instructions. PBMCs from healthy donors were isolated by Ficoll-Paque density gradient from buffy coats from routine blood donations at the Blood Central at Umeå University Hospital, Umeå, Sweden. All cell suspensions except bone marrow aspirates were frozen in fetal bovine serum (FBS) (Gibco) with 10% DMSO and stored in liquid N2. Bone marrow aspirates were obtained from routine sampling at the Department of Pathology, Umeå University Hospital.
4
Single-Cell Isolation and Analysis
5
Multiparametric Flow Cytometry of Murine Splenocytes
6
Spleen Cell Isolation Protocol
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Each excised spleen was pressed and sliced using two slide glasses and then placed onto a 40 μm strainer. Filtrated cells were washed by RPMI-1640 media at 3,000 rpm for 5 min. The supernatant was removed and the cell pellets were treated with lysis buffer (BD PharmLyse Lysis buffer; BD Biosciences, San Diego, CA, USA) at 37°C for 5 min. The lysed cells were rinsed twice with RPMI-1640 media at 1,500 rpm for 5 min. The number of isolated cells was determined by counting with trypan blue stain (Thermo Fisher Scientific Inc., Waltham, MA, USA).
7
Isolation of Infected Macrophages from MRSA-Challenged Mice
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C57BL/6 mice were purchased from Jackson Laboratory and bred onsite to generate animals for experimentation. Age and gender-matched 8–10 week old mice were used. The USA300 MRSA strain was grown overnight in TS broth (37°C, 180 RPM). Bacteria was then subcultured in TSB for 3h at 37°, 180 RPM. Bacteria was washed with PBS and adjusted to 1×10^7 CFU/mL. Three mice were injected intraperitoneally with 300μL. After 1h, cells were collected by peritoneal lavage. Red blood cells were lysed with BD Pharm Lyse Lysis Buffer and cells were stained with antibodies to identify macrophages. Infected macrophages were sorted (SY3200 cell sorter, HAPS1 (100um)) and samples were pooled from the three mice for scRNA-Seq analysis (Figure S6A ). Cells were captured using the 10X Chromium platform and processed according to manufacturer recommendations. All animal studies were performed as per an NYU Grossman School of Medicine Institutional Animal Care and Use Committee (IACUC) approved protocol to the Torres Lab.
8
Comprehensive Tumor Immune Profiling Using Flow Cytometry
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Tumors were excised from mice and peripheral blood was harvested at sacrifice. Tumors were digested using the human Tumor Dissociation Kit (with enzymes H and A only to avoid epitope losses) and the GentelMACS Octo Dissociation with heaters. Cells were then filtered on 70 μm MACS SmartStrainers and washed as per manufacturer’s protocol using RPMI with 10% FBS. Cells were then stained with antibodies for analysis by flow cytometry of tumor infiltrating immune cells (hTIICs) against the following targets: humanCD3-AF700, humanCD33-BV510 and humanCD25-BV711 and mouseCD45-PE/Cy7, humanCD45-BUV395, humanCD19-PE/CF594, humanCD4-BB515, humanCD8-BV421, humanCD14-APC/H7, humanCD56-BV786, humanCD127-BB700, humanPD-1-BUV737 and humanTim3-PE. Blood samples from the same animals were also stained with the same antibody panel before red blood cell lysis using BD Pharm Lyse lysis buffer. All results were acquired using a BD LSRFortessa. Data analysis was done on FlowJo V10. For tSNE dimensionality reduction analysis, tumor and blood immune cells were subsetted and pooled to obtain a representative sample of 20 000 cells. Dimensionality reduction was done using FlowJo’s tSNE implementation and the FlowSOM algorithm (Gassen et al., 2015 (link)) was used to identify clusters in an unbiased manner.
9
Multicolor Flow Cytometry Analysis of Murine B Cell Subsets
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After centrifugation of mice's peripheral blood, red blood cells were lysed using a lysis buffer (BD PharmLyse Lysis Buffer; BD Biosciences, San Diego, CA, USA) to obtain a single-cell suspension. The single-cell suspension was incubated with Purified anti-mouse CD16/32 Antibody (Catalog No. 158002, BioLegend, San Diego, CA, USA) to block nonspecific antibody binding. Subsequently, a cocktail of fluorescence-conjugated antibodies, including CD19, CD138, and PD-L2, was added for staining. Following staining, the cells were fixed with 4% PFA and analyzed using the FACS Celesta™ Flow Cytometer (BD Biosciences, San Diego, CA, USA). The analysis of B cell subsets and the gate scheme in flow cytometry were performed according to previous reports (Fig. 3 A) [23 (link), 24 (link)]. Furthermore, to evaluate cell surface PD-L1 expression, hUC-MSCs were stained with Alexa Fluor 647-conjugated PD-L1 antibody (Additional file 1 : Fig. S1A). All flow cytometry data were analyzed using FlowJo 10.8.1 software (FlowJo, Ashland, OR, USA). For mice lymphocyte-derived single cells, the samples were down-sampled to 30,000 cells per sample. The detailed list of fluorescence-conjugated antibodies used for flow cytometry can be found in Additional file 2 : Table S1.
10
Ethical Isolation and Cryopreservation of PBMCs
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The research was carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Ethical permits were obtained from the Swedish Ethical review authority (No: 2016/53-31, 04-113M, 07-162M and 2014/233) and all samples were collected after receiving informed consent from patient or patient's guardian. Briefly, blood was collected in EDTA tubes and PBMCs were isolated using a Ficoll-Paque density gradient centrifugation. Tonsillar cell suspensions were prepared by tissue homogenizing in RPMI-1640 medium and passed through a 70 µm cell strainer. Red blood cells were lysed using BD PharmLyse lysis buffer according to manufacturer's instructions. PBMCs from healthy donors were isolated by Ficoll-Paque density gradient from buffy coats from routine blood donations at the Blood Central at Umeå University Hospital, Umeå, Sweden. All cell suspensions except bone marrow aspirates were frozen in fetal bovine serum (FBS) (Gibco) with 10% DMSO and stored in liquid N2. Bone marrow aspirates were obtained from routine sampling at the Department of Pathology, Umeå University Hospital.
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